Calcium flux assays are the preferred methods in drug discovery for screening G protein coupled receptors (GPCR). Screen Quest™ Calbryte-520 Probenecid-Free and Wash-Free Calcium Assay Kit provides the most robust homogeneous fluorescence-based assay for detecting the intracellular calcium mobilization. Cells expressing a GPCR of interest that signals through calcium are pre-loaded with our proprietary Calbryte™-520 AM which can cross cell membrane. Calbryte™-520 AM is the brightest calcium indicator available for HTS screening. Once inside the cell, the lipophilic blocking groups of Calbryte™-520 AM are cleaved by non-specific cell esterase, resulting in a negatively charged fluorescent dye that stays inside cells, and its fluorescence is greatly enhanced upon binding to calcium. When cells stimulated with screening compounds, the receptor signals release of intracellular calcium, which greatly increase the fluorescence of Calbryte™-520 AM. The characteristics of its excellent cell retention, high sensitivity, and 100-250 times fluorescence increases (when it forms complexes with calcium) make Calbryte™-520 AM an ideal indicator for measurement of cellular calcium. Calbryte™-520 AM is the only calcium dye that does not require probenecid for better cellular retention. This Screen Quest™ Calbryte-520 Probenecid-Free and Wash-Free Calcium Assay Kit provides the most optimized assay method for monitoring G-protein-coupled receptors (GPCRs) and calcium channels with fragile or difficult cell lines. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation.
1. Prepare cells in growth medium
2. Add Cal-520TM AM dye-loading solution (100 μL/well for 96-well plate or
25 μL/well for 384-well plate)
3. Incubate at room temperature or 37°C for 15-30 minutes
4. Monitor fluorescence at Ex/Em = 490/525 nm
Important Thaw all the kit components at room temperature before use.
Instrument: Fluorescence microplate reader
Excitation: 490 nm
Emission: 525 nm
Cutoff: 515 nm
Recommended plate: Solid black/clear bottom
Other Instruments: FDSS, NOVOStar, FlexStation, ViewLux, IN Cell Analyzer, ArrayScan, FLIPR
Preparation of Stock Solution
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
1. Cal-520TM AM stock solution:
Add 20 μL or 200 μL of DMSO into the vial of Cal-520TM AM (Component A) and mix them well.
Note: 20 μL of Cal-520TM AM stock solution is enough for one plate. Unused Cal-520TM
AM stock solution can be aliquoted and stored at < -20 oC for more than one month if the tubes are sealed tightly.
Note Protect from light and avoid repeated freeze-thaw cycles.
2. Assay buffer (1X):
Mix 9 mL of HHBS (Component C, not included in the kit) with 1 mL of 10X Pluronic® F127 Plus (10X) (Component B) and mix them well.
Preparation of Working Solution
Cal-520TM AM dye-loading solution:
Add 20 μL of Cal-520TM AM stock solution into 10 mL of Assay Buffer (1X) and mix them well.
Note This working solution is stable for at least 2 hours at room temperature.
Note 10 mL dye-loading solution is enough for one 96-wells plate.
Sample Experimental Protocol
1. Add 100 μL/well (96-well plate) or 25 μL/well (384-well plate) of Cal-520TM AM dye-loading solution into the cell plate.
2. Incubate the dye-loading plate in a cell incubator for 30 minutes, and then incubate the plate at room temperature for another 15 - 30 minutes.
Note If the assay requires 37°C, perform the experiment immediately without further room temperature incubation. If the cells can function well at room temperature for longer time, incubate the cell plate at room temperature for 1
hour (It is recommended that the incubation time be no longer than 2 hours.)
3. Prepare the compound plate with HHBS or your desired buffer.
4. Run the calcium flux assay by monitoring the fluorescence intensity at Ex/Em = 490/525 nm.
EXAMPLE DATA ANALYSIS AND FIGURES
The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the baseline corrected values. Then, plot the standards' readings to obtain a standard
curve and equation. This equation can be used to calculate ATP samples. We recommend using the Online Four Parameter Logistics Calculator
Figure 1. Comparison of fluorescent signal response of endogenous P2Y receptor to ATP in CHO-K1 cells. CHO-K1 cells were seeded overnight at 50,000 cells/100 μL/well in a 96-well black wall/clear bottom costar plate. Calcium flux response was
measured with the Cal-520TM AM Probenecid-Free and Wash Free Calcium Assay Kit, FLIPR Calcium 4 Assay Kit, and Fluo-4 Direct Calcium Assay kit. ATP (50 μL/well) was added by FlexStation 3 to achieve the final indicated concentrations.
Assay Genie provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from Assay Genie.